Three-dimensional structured illumination microscopy data of mitochondria and lysosomes in cardiomyoblasts under normal and galactose-adapted conditions

This three-dimensional structured illumination microscopy (3DSIM) dataset was generated to highlight the suitability of 3DSIM to investigate mitochondria-derived vesicles (MDVs) in H9c2 cardiomyoblasts in living or fixed cells. MDVs act as a mitochondria quality control mechanism. The cells were stably expressing the tandem-tag eGFP-mCherry-OMP25-TM (outer mitochondrial membrane) which can be used as a sensor for acidity. A part of the dataset is showing correlative imaging of lysosomes labeled using LysoTracker in fixed and living cells. The cells were cultivated in either normal or glucose-deprived medium containing galactose. The resulting 3DSIM data were of high quality and can be used to undertake a variety of studies. Interestingly, many dynamic tubules derived from mitochondria are visible in the 3DSIM videos under both glucose and galactose-adapted growth conditions. As the raw 3DSIM data, optical parameters, and reconstructed 3DSIM images are provided, the data is especially suitable for use in the development of SIM reconstruction algorithms, bioimage analysis methods, and for biological studies of mitochondria.

The main challenges in SRM beyond the strict sample and imaging system requirements involve phototoxicity, photobleaching, imaging speed, throughput, reconstruction artifacts, data analysis, and the validation of results 4,8 . A major challenge for the developers of reconstruction algorithms and computational methods is the lack of suitable data, as instruments for SRM or the required interdisciplinary knowledge and training necessary for generating good bioimage datasets are to most researchers not readily available. Usually, the complete data following publication related to SRM is not provided 9 . The provision of only compressed illustrations or the lack of metadata are related and important issues hindering the reuse of research data 10 .
The current dataset -outlined in Fig. 1 -was initially acquired to investigate the suitability of 3DSIM for two different biological applications: a) the study of mitochondria-derived vesicles (MDVs) in living or fixed samples, and b) correlative imaging of the tandem-tag acidity sensor mCherry-eGFP with LysoTracker Deep Red (LTDR), both in the cardiomyoblast cell-line H9c2 with a stable expression of mCherry-eGFP fluorescent-tagged trans-membrane domain of the outer mitochondria membrane protein 25 (OMP25). As the mCherry fluorescent protein is acid-stable (pKa < 4.5) while the eGFP is acid-labile (pKa = 6.0) and rapidly quenched in an acidic environment, this tandem-tag construct can be used as an acidity sensor and assay for lysosomal degradation of mitochondria 11 . MDVs act in mitochondria quality control and are small (70-150 nm in diameter), single or double-membrane vesicles that arise through budding from the mitochondria 12 .
Images of cells from two different growth conditions are provided: in normal media containing glucose, or in glucose-free, galactose-adapted growth conditions. Further, two different imaging conditions are provided for both live-and fixed-cell imaging: living cells with or without LTDR staining, and fixed cells chemically preserved using either paraformaldehyde (PFA) or PFA in combination with glutaraldehyde (GA). This extensive and high-quality 3DSIM dataset is provided as a community resource intended for those working on 3DSIM reconstructions, bioimage analysis, or mitochondria research.

Methods
Cell-culture and sample preparation. The rat cardiomyoblast cell-line H9c2 (cells derived from embryonic heart tissue; Sigma Aldrich) was genetically modified using retrovirus to achieve a stable expression of tandem tagged (mCherry-eGFP) mitochondrial outer membrane protein 25 (OMP25)-transmembrane domain (TM). A uniform expression of fluorescence intensity in the cells was achieved through flow cytometry sorting. The stable H9c2 cells were cultured in high glucose (4.5 g/L) Dulbecco's Modified Eagle Medium (DMEM, D5796, Sigma-Aldrich) with 10% FBS, 1% streptomycin/penicillin and 1 µg/ml of puromycin (InvivoGen). For glucose deprivation and adaptation to galactose, the cells were grown in DMEM without glucose (11966-025, Gibco) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM galactose, 10% FBS, 1% streptomycin/ penicillin, and 1 µg/ml of puromycin (InvivoGen). The cells were adapted to galactose for a minimum of 7 days before experiments. The cells were seeded on MatTek dishes (P35G-1.5-14-C, MatTek Corporation) and imaged when they reached approximately 80% confluency.
Labeling of lysosomes (acidic endosomal system) was done by incubating the cells for 40 minutes with 100 nM LysoTracker Deep Red (LTDR, Cat nr L12492, Thermo Fisher) in the normal cell-culture medium (with www.nature.com/scientificdata www.nature.com/scientificdata/ either glucose or galactose). After labeling, the media was replaced with fresh cell-culture media right before live imaging.
Cell fixation. The fixed samples were treated as above, but fixed using either 4% paraformaldehyde (PFA) or 4% PFA + 0.2% glutaraldehyde (GA) in phosphate-buffered saline (PBS, preheated to 37°C) for about 30 min at room temperature. The samples were then washed and re-immersed in PBS before imaging. Only the GA samples were labeled using LTDR.
Imaging conditions. The living cells were imaged in their usual growth medium at 37°C with atmospheric gas levels. The fixed samples were imaged at either room temperature or at 37°C in PBS.
Microscope. The images were acquired using a DeltaVision OMX V4 Blaze imaging system (GE Healthcare) equipped with a 60X 1.42NA oil-immersion objective (Olympus), three sCMOS cameras, and 405, 488, 568, and 642 nm lasers for excitation. The vendor-specified optical resolution of the 3DSIM system is 110-160 nm laterally, and 340-380 nm axially, depending on color channel. To surpass the diffraction limit, this SIM set-up uses sinusoidal illumination patterns and acquires 120 images per 1 µm z-stack thickness (3 illumination angles times 5 phase shifts times 8 planes/µm thickness) per color channel. Super-resolution 3D images are then obtained via image processing using the reconstruction software described under Image processing. Image processing. Image reconstruction and channel registration. Image deconvolution and 3DSIM reconstructions were completed using the manufacturer-supplied softWoRx program (GE Healthcare). Image registration (color channel alignment) was also performed in the same program using experimentally-measured calibration values compensating for minor lateral and axial shifts, rotation, and magnification differences between cameras. The pixel area of the 3DSIM images is 1/4 of the acquired raw data (40 nm by 40 nm vs. 80 nm by 80 nm). The files in the SIMmaxPRj folders were additionally maximum intensity z-projected as a final step.
Image analysis and processing. Image analysis and processing beyond the preprocessing described above was done using Fiji 13 . This includes the conversion from DV to TIFF image files (preserving bit-depth and metadata), the generation of AVI movies (maximum intensity z-projected and bleach corrected image sequenced using the exponential fit option), and visualization using orthogonal views.

Data records
The data is published on the DataverseNO repository in the UiT Open Research Data collection https://doi. org/10.18710/PDCLAS 14 . The image files were originally collected as DV files but were losslessly (preserving bit depth and metadata) converted to TIFF (either 16-or 32-bit hyperstacks) before archiving. For each collected image file or image reconstruction, there is an associated text file describing the acquisition parameters. Records of the image acquisition are also available in the image metadata (in ImageJ/Fiji, the metadata can be read using the keyboard shortcut "I").
In addition to a description of the overall dataset and folders, an explanation of how the file naming convention translates back to experimental conditions is available in the ReadMe file associated with the published dataset.
To achieve a successful upload to the data repository, the data was stored (losslessly) in ZIP archives. A complete list of the files contained within the different ZIP files is provided in Documentation/00_FileOrganization. txt.
Data folders and experimental conditions. The data is collected into four main imaging experiments as summarized in Table 1. Each of the main folders contains images of cells from both the GAL and GLU growth conditions (indicated in the file names): • The Fixed/PFA_eGFP data is of cells fixed using 4% PFA in PBS. The data was found non-suitable for MDV quantification due to the fragmentation of mitochondria. Most of the images are of the eGFP channel only. • The Fixed/GA_LTDR-mCherry-eGFP data are samples prepared the same way as for Live-cell/_LTDR, but fixed using 4% PFA + 0.2% GA in PBS. The mitochondria no longer appear fragmented as for Fixed/PFAe_ eGFP, but with a higher autofluorescence due to the GA treatment, which can cause some additional SIM reconstruction artifacts. www.nature.com/scientificdata www.nature.com/scientificdata/

Technical Validation
Imaging system and data acquisition. The commercial OMX Blaze 3DSIM imaging system was installed and quality checked by a system engineer. The image acquisition, optimization of immersion oil and reconstruction quality was overseen by an expert user. Further, and according to individual needs, both raw and reconstructed 3DSIM data can be checked for particular features and quality measures using the open-source Fiji plugin SIMcheck 15 . The main characteristics of good SIM raw data are outlined in Fig. 2.
Image quality. Although of overall good quality, SIM images can never be assumed to be completely without reconstruction artifacts. Due to cell and sample variability, some variation in image quality is also expected, even for the same sample and identical acquisition parameters. When the SIM artifacts are of significantly lower intensity values than the biological features of interest, such that the artifacts can be removed by linearly adjusting the image brightness, we consider the image quality good. The nonphysical negative intensity values resulting from the reconstruction algorithm can usually be discarded without losing biological context. This is illustrated in Fig. 3. Recognizing what are SIM artifacts or actual sample features can require some training. Common artifacts, their causes together with different trade-offs in SIM acquisition are explained by Demmerle et al. 16 . Sample quality after aldehyde fixation. The main goal of sample fixation in microscopy is to provide a snapshot of the sample morphology without the effects of motion blur or phototoxicity during imaging, cellular changes due to environmental changes or just the duration of time between sample preparation and imaging. www.nature.com/scientificdata www.nature.com/scientificdata/ What is a suitable fixation protocol depends on the sample as well as the imaging method and desired analysis 17,18 . For our cells with mitochondria-derived (nano-) vesicles as the target of our study, we first opted for fixation using 4% PFA in PBS. At 3DSIM resolution and with the desire of quantifying nanovesicles, this fixation method was deemed unsuitable for organelle preservation because of clear fragmentation of mitochondria obstructing reliable quantification of small vesicles. Using instead a combination of PFA and GA preserved the live-cell morphology much better. This is nicely illustrated in Fig. 2 of reference 19 using the same dataset. It also shows a comparison of a conventional (deconvolved) image, where the difference in live vs. fixed cell morphology is much harder to detect and thus might be less significant for other types of imaging techniques and analysis. A downside of the GA-based fixation is that GA itself exhibits a strong fluorescence 20 . This causes unspecific fluorescence which can affect the SIM reconstruction quality. We found 0.2% GA together with 4% PFA to be a suitable compromise for our application.

Cell-line.
Biological context. Glucose serves as the primary metabolic fuel of mammals 21 . Glucose is converted to energy in the form of adenosine triphosphate (ATP) in a process termed glycolysis. The main ATP energy production in normal cells however, is conducted in the mitochondria through oxidative phosphorylation (OXPHOS). H9c2 cells under normal culture conditions with high glucose levels primarily utilize glycolysis for ATP generation. In contrast, H9c2 cells deprived of glucose and adapted to galactose rely entirely on OXPHOS to meet their energy requirements. As a consequence, glucose deprivation has been shown to render the cells more sensitive to mitochondrial damage 22 . This is likely to cause differences between cells under the two different cultivation conditions, although we have noted also a large variation within the two groups when it comes to e.g., mitochondrial morphology, amount of lysosomes, and amount of mitochondrial fragments found in lysosomes. In reference 19 , the glucose-deprived cells were shown to contain more mitochondria-derived vesicles than the cells given glucose (i.e., normal growth conditions).

Usage Notes
The images (TIFF and OTF files) can be opened in ImageJ/Fiji or other programs for image processing. Please be aware of the negative intensities in the 3DSIM reconstruction. The negative intensities are a peculiarity often resulting from the SIM reconstruction process. Although an undesirable image artifact, keeping the negative intensities can be important for checking the reconstruction quality of SIM data 15 and for recognizing artifacts by visual inspection 16 . For most purposes, the negative intensities can be set to zero for visualization and before analysis (e.g. in Fiji, using the Brightness and Contrast tool). The log or image registration files (txt or dat) can be opened using any text editor.
An overview of the image content, quality and biological events of the larger 3DSIM time-lapse files of LiveCell can be quickly accessed through the AVI_movies folder. Here, events like extending mitochondria-derived tubules or MDV dynamics can be observed. Similarly, the files in SIMmaxPrj can also be used as a single-plane visualization tool to get an overview of the data without downloading or opening the entire 3DSIM files. The Conventional folders contain conventional fluorescence microscopy data, deconvolved images, and transmission microscopy images as a reference for the super-resolution microscopy data. Images depicting special cases of mitochondria away from their normal confinement inside cells are provided by 20210511_H9c2-dTag_GAL_PFA4-GA02_LTDR100nm-40m_1520_con_001 (conventional image in Fixed/ GA_LTDR-mCherry-eGFP/GAL) and 20210511_H9c2-dTag_GAL_LTDR100nm-40m_1520_sim256_013 in LiveCell/LTDR/GAL (challenging 3DSIM data). These files should be discarded from analysis concerning, e.g., the number or morphology of mitochondria and lysosomes inside cells. www.nature.com/scientificdata www.nature.com/scientificdata/ The experimental meaning of folder and file names are explained in the dataset's ReadMe file (in the DATA & FILE OVERVIEW section). The experimental settings for each image file can be accessed without opening the images via the text files in the RawSIM_log folders. Details of the 3DSIM reconstructions conducted are available in the reconstruction log files in the 3DSIM/SIRlog folder. The parameter files used for the 3DSIM reconstructions and for the channel registration/alignment by the commercial softWoRx program are provided in the Parameterfiles folders. These contain experimental point spread functions (PSFs, OTF files) for three different wavelengths (green, red, and far red channels) and image registration data (dat file). The point spread functions describe the imaging systems' response to a point emitter, or for this experimental case, to a 100 nm fluorescent bead (of different emission wavelengths).
The LiveCell/LTDR and Fixed/GA_LTDR-mCherry-eGFP data are best suited for comparing the GLU and GAL growth conditions. Note, however, the rapid photobleaching of LTDR.
Apart from biological inferences, the long time-lapses of the Live-cell data might be interesting 3DSIM data for testing the performance of new reconstruction algorithms, as the signal-to-noise ratio (and the 3DSIM image quality) goes down towards the end. Acquisition speed. The additional filename annotations fast, faster, fasterer for the LiveCell/eGFP experiments indicate changes in the acquisition settings achieving gradually faster acquisition rate, i.e., changes in camera mode, camera read area, exposure time, stack size and time-lapse (i.e., additional time-delay added between each time-point other than single-image acquisition time). Only one setting was changed at the time such that the effect could be readily assessed by comparing different experiments (i.e., image files). The achieved acquisition rate for the LiveCell/eGFP experiments is in the range 1 s -5 s per 3DSIM volume. For the particular example displayed in Fig. 1 19 , the 3DSIM frame rate was 1.5 s (file: 20210420_H9C2-dTag_GAL_37C_1520_sim-fasterer_018_SIR_PRJ).
The acquisition settings are most reliably read from the associated image log files (e.g. in LiveCell/eGFP/ RawSIM/log), but can in some cases be found in the image metadata as well. The actual resulting acquisition time for an image frame (which can be significantly different from the targeted Time-lapse setting) can be read from the image metadata as timestamps. For multi-channel data, W0, W1, W2, refer to the acquisition of three different color channels (e.g., Cy5, A568, GFP), which have different timestamps as sequential acquisition mode was used. The example Extended header Z0 W2 T0:timeStampSeconds = 5.143928, means that this was the first z-plane in the image stack, the third color channel, and the first time-point acquired after 5.15 seconds.
Tests of acquisition speed and parameters were also conducted for the LiveCell/LTDR multi-channel experiments. The first images of GLU (name containing 002, 003, 004, 005) are acquired using different parameters than the rest. 002 and 003 were acquired using higher illumination intensities for the remaining data (30% transmission (%T) rather than 10%T for the GFP and A568 channels, see the acquisition log files). 004 is only one frame, 005 differs from the rest (006-016 in GLU and 003-012 in GAL) only by Time-lapse being set to 3 s instead of 5 s, but resulting in about the same recorded time (70.5 s for 10 three-channel 3DSIM volumes) as the microscope cannot image any faster with the experimental settings given. For the file 017 in the same folder, the time-lapse settings were 10 s. Here, the time between each recorded time-point is 10 s.

Code availability
The 3DSIM reconstructions, image deconvolutions and associated image registrations were completed using the proprietary software softWoRx 7.0.0 following the microscope.

Funding
Open Access funding provided by UiT The Arctic University of Norway.